virology assays
Recorded terms: quantitative PCR; quantitative RT-PCR; real-time quantitative RT-PCR; RT-PCR; RT-qPCR
The Capicua and ATXN1L repressor complex binds an eight-nucleotide motif at interferon and interferon-stimulated gene loci to hold them repressed during homeostasis in human and mouse cells, and is degraded through EGFR-MAPK signaling early in respiratory viral entry, which relieves that repression.
Archaeal members of the L30 protein family, but not their orthologues from any other domain of life, block production of the spliced influenza A virus, influenza B virus and isavirus transcripts without measurably disturbing host splicing, identifying a shared and apparently noncanonical feature of orthomyxovirus splicing.
In golden hamsters, productive SARS-CoV-2 replication in the airways generates circulating type I and III interferon that primes every organ against infection, and blunting or bypassing that airway response permits viremia and productive infection of liver, kidney, spleen and brain.
Intranasal SARS-CoV-2 infection of golden hamsters deposits viral RNA without infectious virus in dorsal root ganglia and spinal cord, producing a milder but longer-lasting mechanical hypersensitivity than influenza A virus and a neuropathic dorsal root ganglion transcriptome at 31 days, from which ILF3 emerges as an analgesic target validated in mouse pain models.
Stress granules restrain rather than amplify double-stranded RNA sensing, and cells lacking the granule nucleators G3BP1 and G3BP2, UBAP2L or PKR respond to double-stranded RNA with excessive RIG-I-like receptor, PKR and OAS activation and MAVS-dependent apoptosis.
Kinase substrate specificity profiling assigns the phosphorylation cluster in the SARS-CoV-2 nucleocapsid SR-rich domain to a cascade initiated by SRPK1 and SRPK2 and extended by GSK-3 and casein kinase 1, whose inhibition suppresses coronavirus replication.
Benchmarked against pandemic influenza in golden hamsters, SARS-CoV-2 uniquely sustains interferon signaling, chemokine production and myeloid activation in olfactory bulb and epithelium a month after clearance, alongside altered behavior and matching signatures in recovered human olfactory tissue.
In golden hamsters, influenza A virus infection reduces SARS-CoV-2 replication during coinfection, after preinfection, and even one to two weeks after influenza has been cleared, while SARS-CoV-2 leaves influenza replication in vivo unchanged.
A microfluidic bronchial airway chip lined with differentiated human airway epithelium and pulmonary endothelium reproduces strain-dependent influenza virulence, cytokine output and neutrophil recruitment, and when drugs are delivered at clinically achievable blood concentrations under flow it separates candidates that work in cell lines from those that also work in hamsters challenged with SARS-CoV-2.
A genome-scale CRISPR loss-of-function screen in ACE2-expressing human alveolar epithelial cells ranks every protein-coding gene by the effect of its loss on SARS-CoV-2 infection, converging on endosomal machinery, and links several top hits to increased cholesterol biosynthesis and, for RAB7A, to intracellular sequestration of ACE2.
Longitudinal tracking of antigen-specific lymphocytes in golden hamsters shows that memory from the founder strain of SARS-CoV-2 clears a beta variant rechallenge and that transferred memory T cells alone lower viral load, yet protected animals still infected every cohoused naive partner.
Longitudinal transcriptional and histological profiling of SARS-CoV-2 infected golden hamsters maps a wave of inflammation that reaches tissues with little or no productive replication, and shows that intranasal type I interferon given before or after challenge lowers viral load and disease burden.
Human ocular surface tissue carries SARS-CoV-2 entry machinery and supports productive replication, with the limbus most permissive in both adult donor cells and stem cell derived whole-eye cultures, where infection drives NF-kB chemokine induction and blunted interferon signaling.
SARS-CoV-2 infection of human lung epithelial cells engages NF-κB at chromatin, transcriptional, protein and post-translational levels without engaging the type I interferon transcription factors, and loss of p65 or p50 abolishes viral replication in a manner rescued by reconstituting RelA transcriptional activity.
Isolating the SARS-CoV-2 Spike D614G substitution from the ORF1b P314L variant it travels with, through pseudotyped lentiviral particles and a trans-complementation assay with replication-competent virus, shows that the single Spike change raises entry efficiency across human lung, liver and colon cell lines without altering S1 affinity for ACE2.
A panel of eight human pluripotent stem cell derivatives spanning all three germ layers, together with adult primary islets and liver organoids, identifies pancreatic alpha and beta cells, hepatocytes, cholangiocytes, cardiomyocytes and dopaminergic neurons as permissive to SARS-CoV-2 and shows that permissiveness does not track ACE2 expression alone.
Across cell lines, primary bronchial epithelium, ferrets and patient material, SARS-CoV-2 infection produces a transcriptional response distinguishable from that to other respiratory viruses, combining low type I and type III interferon induction with a moderate interferon-stimulated gene response and strong chemokine and IL-6 expression.
A time-resolved phosphoproteomic survey of SARS-CoV-2-infected cells showing that infection acts mainly through signalling rather than protein abundance, activating casein kinase II and the p38 cascade while shutting down mitotic kinases, and converting that kinase profile into inhibitors with antiviral activity.
Forcing an interferon-stimulated gene program in human induced pluripotent stem cells with a constitutively active IRF7 produces lasting transcriptional change and impaired germ layer differentiation, supporting the proposal that the canonical type I interferon system and pluripotency are difficult to hold simultaneously.
A survival-based genome-wide CRISPR knockout screen in human lung epithelial cells selected with an avian H5N1 isolate recovers sialic acid biosynthesis and transport as the dominant requirement for influenza entry, with the CMP-sialic acid transporter SLC35A1 as the top hit, and identifies the transcriptional repressor capicua as a negative regulator of cell-intrinsic immunity.
A one-step recombineering strategy that inserts hematopoietic-specific miR-142 target sites into the untranslated region of the human cytomegalovirus IE2 transcript permits virus rescue in fibroblasts while silencing IE2 selectively in myeloid cells, revealing that IE2 loss raises rather than abolishes replication in macrophages.
An attenuated influenza A virus engineered to deliver individual artificial small interfering RNAs enables a fitness-based loss-of-function screen inside an infected mouse, and that screen identifies MDA5 as a contributor to the antiviral response to influenza A virus despite the established role of RIG-I as the sensor that induces interferon beta.
Rapid vector-delivered destruction of the entire cellular microRNA population shows that post-transcriptional silencing makes no measurable contribution to the acute transcriptional response to double-stranded RNA or to type I interferon, while sustained loss of microRNAs derepresses a broad set of chemokines and proinflammatory cytokines in fibroblasts and in mouse lung.
Replication-incompetent influenza-based vectors carrying no DNA intermediate deliver coding messages and functional small RNAs together to primary human and mouse cells and to mouse lung, with output and cytotoxicity tuned by microRNA target sites in the nucleoprotein segment.
A Cre recombinase-expressing influenza A virus combined with Cre-responsive reporter and ablation mouse strains showed that a subpopulation of directly infected lung cells, predominantly club cells, survives productive infection, sustains elevated interferon-stimulated gene and chemokine expression, and contributes to bronchiolar epithelial damage after virus is cleared.
Sustained IRF7 activity induces the kinase MAP3K8, which phosphorylates the proline-rich hinge of IRF3 and redirects it from homodimers into IRF3 and IRF7 heterodimers, broadening the antiviral transcriptome and scaling the cellular response to the persistence of the viral threat.
Engineering vesicular stomatitis virus to eliminate RISC-loaded small RNAs attenuates rather than enhances replication in mice, and confers no replication advantage even when interferon signaling is removed, arguing that small RNA silencing does not contribute to mammalian antiviral defense.
Engineering target sites for miR-192, a microRNA abundant in human and mouse respiratory tissue but absent from the ferret respiratory tract, into the influenza A virus hemagglutinin segment attenuates the virus in mice while leaving replication and transmission in ferrets intact, providing a genetic layer of biocontainment for transmission studies.
Influenza A virus engineered to be silenced by the hematopoietic microRNA miR-142 shows that replication inside antigen presenting cells is dispensable for CD8 T cell priming and clearance but required for much of the RIG-I-dependent type I interferon response in vivo.
A negative-sense cytoplasmic RNA virus, vesicular stomatitis virus, can be engineered to produce mature Dicer-dependent miR-124 that loads into Argonaute 2, silences targets, reaches many tissues in mice, and persists after the vector itself is cleared.
Inserting hematopoietic-specific miR-142 target sites into the dengue virus 3-prime untranslated region excludes replication from macrophages and dendritic cells in vivo and abolishes spread to other tissues, identifying hematopoietic cells as the dominant amplification compartment.
Phosphorylation of STAT1 serine 708 by IKKε blocks formation of the STAT1 homodimer that constitutes GAF while leaving the STAT1 and STAT2 heterodimer of ISGF3 intact, biasing the shared STAT1 pool and the interferon-stimulated transcriptome toward the type I response.
Influenza A virus produces a family of 22 to 27 nucleotide small viral RNAs corresponding to the 5 prime end of each genomic segment, which accumulate as the polymerase shifts toward genome synthesis and whose inhibition selectively depletes genomic RNA without comparably affecting messenger or complementary RNA.
IRF7 and ISGF3 engage overlapping interferon-stimulated response elements and drive largely overlapping antiviral transcriptomes, so that a substantial interferon-like gene program is still induced when type I and type III interferon signaling are both absent.
Engineering microRNA response elements for a mammalian-ubiquitous microRNA directly into the influenza A virus nucleoprotein coding sequence attenuates the virus in mice while leaving growth in embryonated chicken eggs intact, yielding live attenuated vaccine candidates for H1N1 and H5N1.
Mice lacking IKKε produce normal interferon-β but fail to induce roughly a third of interferon-stimulated genes, because interferon activates IKKε, which phosphorylates STAT1 at Ser708 and thereby determines whether ISGF3 occupies a subset of response elements.